Apolipoprotein B (apo B) is a large hydrophobic protein synthesized in the liver and small intestine of mammals. Apo B serves an essential although incompletely understood role in the assembly and secretion of triglyceride-rich lipoproteins (chylomicrons and very low density lipoproteins) and also functions in the catabolic clearance of low density lipoprotein (LDL), the major transport vehicle of plasma cholesterol of humans.
Mammalian apo B is the product of a single gene which maps to the p23-p24 region of chromosome 2 (Chan, et al. 1985; Mehrabian, et al. 1986; Luo, et al. 1986; Li, et al., 1988). Apo B RNA is expressed and processed in a tissue-specific fashion. One form of apo B is synthesized in the human liver as a protein of 4536 amino acids with a relative molecular mass of about 5 12,000 daltons. This form of the protein is referred to on a centile scale as apo B100 (Knott, et al. 1986; Yang, et al. 1986; Chen, et al. 1986; Cladaras, et al. 1986; Ludwig, et al. 1987). Several important structural domains have been identified for apo B 100 based upon cDNA sequence and monoclonal epitope mapping. The most important of these are the low density lipoprotein (LDL) receptor binding domain and the attachment site of apolipoprotein (a) which both reside in the carboxyl terminal half of the protein (Yang, et al. 1990; Law and Scott 1990; Pease, et al. 1990).
By contrast, the intestinal form of the protein contains about 2152 amino acids, is colinear with the amino terminal half of apo B100, and is referred to as apo B48 (Innerarity, et al. 1987; Hardman, et al. 1987). Apo B48 is found in the systemic circulation in association with intestinally derived lipoprotein particles, namely chylomicrons and chylomicron remnants. Those particles are cleared from the plasma compartment via a principally hepatic receptor that has been partially characterized and is referred to as the low density lipoprotein-receptor related protein or LRP (Herz, et al. 1988; Kowal, et al. 1989). That receptor recognizes apolipoprotein E as its major ligand (Beisegel, et al. 1989).
The biological relevance of apo B48 likely relates to the absence of the apo B100 carboxyl domains. Lipoprotein particles containing apo B48 have a catabolic fate different from those with apo B100 which are cleared principally via the LDL-receptor. The implications for this observation in terms of atherosclerosis susceptibility remain to be tested experimentally but it suggests several models whereby such characteristics of an intestinal particle may have evolved to facilitate their function as an efficient delivery system for dietary triglyceride. By contrast, the finer regulation of plasma cholesterol homeostasis may be achieved through hepatic very low density lipoprotein (VLDL) secretion and ultimately LDL uptake.
The post-transcriptional modification of apo B RNA is referred to as apo B RNA editing. Editing is distinct from other co- or post-transcriptional processing events such as capping, polyadenylation and splicing. Apo B RNA editing represents a departure from one of the central tenets of molecular biology that DNA encodes an RNA template which is identical and which subsequently specifies a predictable protein. Several examples of RNA editing have been described in lower eukaryotes and classified according to the underlying mechanism (Cattaneo 1991; Soliner-Webb 1991) such as post-transcriptional insertion or deletion of uridine residues in trypanosome mitochondrial genes which results in the production of a translationally competent open reading frame. Other forms of RNA editing include the post-transcriptional insertion of guanosine residues in paramyxovirus and cytidine residues in physarum polycephalum.
Apo B RNA editing was described in 1987 when several groups simultaneously reported the site-specific modification of apo B RNA as the underlying mechanism for the production of distinct isoforms of apo B from the human liver and small intestine (Powell, et al. 1987; Chen, et al. 1987; Hospattankar, et al. 1987). Those studies demonstrated that nucleotide 6666 in human and rabbit intestinal apo B cDNA was changed from the genomically templated cytidine to a uridine residue. That change modified codon 2153 from a CAA which encodes glutamine to a UAA which encodes an in-frame stop codon. Those findings indicated that intestinal apo B was likely the product of a single apo B gene in which codon 2153 was altered to produce a translational stop codon and thereby specify a truncated apo B (apo B48) as the primary translation product.
Apo B RNA editing in the mammalian enterocyte occurs as a developmentally regulated event in human (Teng, et al. 1990), rat (Wu, et al. 1990) and pig small intestine (Teng, et al. 1990). Teng et al. demonstrated that human intestinal apo B was more than 90% unedited in fetal small intestinal RNA from late first trimester samples. Teng et al. also demonstrated that the early gestation fetal small intestine synthesizes and secretes both apo B100 and apo B48, indicating that the unedited form of the apo B transcript is translationally competent in the small intestine and, furthermore, leads to apo B100 secretion. As the small intestine undergoes morphological maturation during the second trimester, the proportion of edited apo B RNA increases such that at 19-20 weeks gestation, small intestinal apo B RNA is approximately 80-90% edited. Adult small intestine was found to contain a variable quantity of unedited apo B RNA, varying from 3-19% in one series (Teng et al., 1990).
Rat apo B RNA editing is developmentally regulated in both the small intestine and liver. Wu et al. demonstrated that the temporal sequence of the developmental changes in apo B RNA editing was distinct for the liver and small intestine with a striking increase in intestinal editing prenatally while the hepatic transcript was largely unedited until postnatal day 20. Coleman et al. demonstrated that coordinate changes in rat hepatic triglyceride metabolism occurred over this same time period showing that the emergence of edited apo B RNA in both the rat liver and intestine coincides with developmental changes in triglyceride metabolism (Coleman et al., 1988).
A human colon cancer-derived cell line (Caco-2) which, in culture, undergoes a form of "spontaneous differentiation" and displays certain phenotypic characteristics of developing enterocytes has been used to study apo B RNA editing. During the course of differentiation from pre- to late postconfluency, apo B RNA abundance increased 20-fold but the proportions of edited to unedited transcript remained unaltered at less than 5% at all times studied (Teng et al., 1990). Thus, in this cell line apo B RNA abundance appears to be regulated by mechanisms distinct from those which influence apo B RNA editing. Other investigators (Jiao, et al. 1990) using this cell line have found that apo B RNA editing increases when the cells are grown on semipermeable filters rather than plastic.
Mammalian apo B is expressed in a tissue-specific manner with gene transcription and protein synthesis predominantly confined to the adult liver and small intestine. In the fetus, however, apo B RNA and protein biosynthesis occurs in a number of extraintestinal, extrahepatic sites. In the rat (Wu, et al., 1990; Demmer, et al. 1986) such tissues include the placenta and fetal membranes while in humans, such tissues include lung, kidney, stomach, colon, adrenal and fetal membranes (Teng, et al 1990; Hopkins, et al. 1987). Analysis of PCR amplified cDNA samples indicated that apo B cDNA was edited to a varying extent (10-50%) in all the fetal tissues examined with the notable exception of the liver and in all those tissues, apo B cDNA was edited to a progressively greater extent during development. Those fetal tissues synthesize and secrete apo B100 but not apo B48, thus, demonstrating that apo B mRNA is translationally active in these locations.
Thyroid hormone modulates hepatic apo B RNA editing in the adult rat (Davidson, et al. 1990). Several genes respond to both T3 and high carbohydrate intake (Davidson, et al. 1990). Baum et al. studied groups of rats fasted for 24 to 48 hours and other groups fasted for 48 hours and subsequently fed a high carbohydrate, fat-free diet for 24 or 48 hours (Baum et al., 1990). This maneuver produced a 30-fold range of hepatic triglyceride concentration from a nadir at 48 hours fasting to a peak at 48 hours refeeding a high carbohydrate diet. In association with these changes, apo B synthesis rates were determined following intraportal vein administration of tritiated leucine and quantitative immunoprecipitation of apo B. In animals fasted for 48 hours there was a decrease in the ratio of apo B48 to apo B100 synthesis and a corresponding decrease in hepatic apo B RNA editing. In animals fasted for 48 hours and the refed a high carbohydrate diet for either 24 to 48 hours, there was no apo B100 synthesis detectable and hepatic apo B RNA was greater than 90% edited. In association with these findings, serum apo B isomorphs as demonstrated on Western blots, were found to be altered in parallel such that control animals and animals fasted for 24 to 48 hours demonstrated mostly apo B100 in their serum while animals fasted and refed a high carbohydrate diet demonstrated essentially only apo B48. Apo A-1 and A-IV RNA abundance and protein biosynthesis were also increased approximately 2-4-fold in the animals refed a high carbohydrate diet for 48 hours. Those changes are thus of a lesser magnitude but in the same direction as encountered (Davidson, et al. 1988) with T3 treatment.
Glickman has shown that the administration of ethinyl estradiol to rats for five days produced a decrease in hepatic but not small intestinal apo B RNA editing and a corresponding increase in the relative synthesis rates of apo B100 (Seishima, et al. 1991). Those findings show a strong correlation between hepatic apo B RNA editing and apo B100 synthesis. A decrease in hepatic apo B RNA editing was associated with uncontrolled non-insulin dependent diabetes mellitus (Jiao, et al. 1991).